Work from our group and others has demonstrated that DNase II enzymes are essential for degradation of ingested cellular DNA after phagocytosis. The cloning of the Drosophila dnase II gene by my group has allowed us to pursue additional questions related to the biological function of this enzyme. Using RNA interference and available DNase II-deficient lines, we have demonstrated that DNase II-deficiency results in increased susceptibility to bacterial infection. To determine if DNase II- deficient flies were defective in other processes such as engulfment, antimicrobial response, etc., we performed a series of genome-wide expression microarray analyses of infected and uninfected control and DNase II-deficient flies. One unexpected result of these array analyses was the detection of a normal antimicrobial peptide (AMP) response after bacterial infection that should have protected the infected flies from infection. Due to the apparently normal humoral and cellular response of DNase II-deficient flies, the misregulation of other genes was analyzed. Our microarray results revealed that the expression of a significant number of genes (n=42 genes, p=0.05) was affected by the DNase II-depletion prior to infection. Interestingly, one of the highest up-regulated open reading frames (CG33346) in DNase II-deficient flies was found to encode a novel non-specific endonuclease. This endonuclease, which we have named DNase III, belongs to an evolutionarily conserved family of nucleases that includes the mammalian Endo-G protein involved in Caspase- independent DNA degradation. The DNase III gene was subsequently cloned and introduced into a prokaryotic expression vector and found to encode a bona fide cation-dependent endonuclease. Interestingly, a second highly homologous ORF, CG9989 (DNase III), was found adjacent to the CG33346 gene that was detected in prior microarray analyses as a gene up-regulated by bacterial infection and parasitic wasp invasion. Our recent array analyses have revealed that expression of CG9989 is up-regulated by 2-3 fold only after infection with Gram positive but not by Gram negative bacteria. Our data indicate that the novel DNase genes are differentially up-regulated by infection. In this proposal, we intend to determine the function of DNase III enzymes primarily via RNAi- mediated gene depletion. These experiments are also designed to determine if DNase III is up- regulated to compensate for the loss of DNase II expression. PUBLIC HEALTH RELEVANCE: Two novel related DNA degradation enzymes were discovered whose genes are activated by distinct stimuli. One of these genes is activated when another nuclease is absent and the other when flies are infected by bacteria. Apart from the characterization of the two novel nucleases, the proposed work can also lead to the discovery of a novel DNA sensing mechanism in fruit flies and perhaps other organisms.